Aaron B. Baker

Natural History of Experimental Coronary Atherosclerosis and Vascular Remodeling in Relation to Endothelial Shear Stress. A Serial, In Vivo Intravascular Ultrasound Study

Background— The natural history of heterogeneous atherosclerotic plaques and the role of local hemodynamic factors throughout their development are unknown. We performed a serial study to assess the role of endothelial shear stress (ESS) and vascular remodeling in the natural history of coronary atherosclerosis. Methods and Results— Intravascular ultrasound–based 3-dimensional reconstruction of all major coronary arteries (n=15) was performed serially in vivo in 5 swine 4, 11, 16, 23, and 36 weeks after induction of diabetes mellitus and hyperlipidemia. The reconstructed arteries were divided into 3-mm-long segments (n=304). ESS was calculated in all segments at all time points through the use of computational fluid dynamics. Vascular remodeling was assessed at each time point in all segments containing significant plaque, defined as maximal intima-media thickness ≥0.5 mm, at week 36 (n=220). Plaque started to develop at week 11 and progressively advanced toward heterogeneous, multifocal lesions at all subsequent time points. Low ESS promoted the initiation and subsequent progression of plaques. The local remodeling response changed substantially over time and determined future plaque evolution. Excessive expansive remodeling developed in regions of very low ESS, further exacerbated the low ESS, and was associated with the most marked plaque progression. The combined assessment of ESS, remodeling, and plaque severity enabled the early identification of plaques that evolved to high-risk lesions at week 36. Conclusions— The synergistic effect of local ESS and the remodeling response to plaque formation determine the natural history of individual lesions. Combined in vivo assessment of ESS and remodeling may predict the focal formation of high-risk coronary plaque.

The role of low endothelial shear stress in the conversion of atherosclerotic lesions from stable to unstable plaque.

Purpose of review Local hemodynamic factors are major determinants of the natural history of individual atherosclerotic plaque progression in coronary arteries. The purpose of this review is to summarize the role of low endothelial shear stress (ESS) in the transition of early, stable plaques to high-risk atherosclerotic lesions. Recent findings Low ESS regulates multiple pathways within the atherosclerotic lesion, resulting in intense vascular inflammation, progressive lipid accumulation, and formation and expansion of a necrotic core. Upregulation of matrix-degrading proteases promotes thinning of the fibrous cap, severe internal elastic lamina fragmentation, and extracellular matrix remodeling. In the setting of plaque-induced changes of the local ESS, coronary regions persistently exposed to very low ESS develop excessive expansive remodeling, which further exacerbates the proinflammatory low ESS stimulus. Recent studies suggest that the effect of recognized cardioprotective medications may be mediated by attenuation of the proinflammatory effect of the low ESS environment in which a plaque develops. Summary Low ESS determines the severity of vascular inflammation, the status of the extracellular matrix, and the nature of wall remodeling, all of which synergistically promote the transition of stable lesions to thin cap fibroatheromata that may rupture with subsequent formation of an occlusive thrombus and result in an acute coronary syndrome.

Aldehyde-amine chemistry enables modulated biosealants with tissue-specific adhesion.

Soft-tissue surgical sealants provide an ideal material class for assessment of tissue–material interactions. Sealant adhesion can be rigorously quantified through a series of functional assays that supplement characterizations of tissue reactivity and material fate. A collection of experimental techniques can be exploited to elucidate mechanistic aspects of tissue–material interactions with general implications, extending beyond the immediate scope of adhesive materials. Moreover, though sealants are routinely used in clinical procedures, active questions and limitations force physicians to choose between extremes of adhesion strength and biocompatibility. [1] Common cyanoacrylate derivatives adhere strongly to tissue, but their vigorous and uncontrolled tissue crosslinking along with the release of toxic degradation by-products dramatically impedes healing and regeneration processes. [2] The polymerization of alkylcyanoacrylates occurs via anionic and zwitterionic polymerizations in the presence of weak bases such as alcohols, water, and amino acids encountered in living tissues. [3] Cyanoacrylates with short side alkyl chains (methyl or ethyl) rapidly degrade to form cyanoacetate and formaldehyde, characterized by acute and chronic inflammation. The longer alkyl chains degrade slower, resulting in more limited accumulation of toxic byproducts that may be effectively eliminated by tissues. Histotoxicity depends on the vascularity of tissues, being greater in well-vascularized soft tissues. [4] Fibrin glues represent the opposite polar extreme along the spectrum of sealants [4] eliciting a mild tissue response, but with relatively non-specific and minimally adhesive tissue interaction. [5–7] Though these and all sealants rely on intimate tissue–material interactions for functional adhesion, target-tissue properties have been largely ignored in material design. Instead, one general formulation is proposed for application to the full range of soft tissues across diverse clinical applications. [8–13] Here, we demonstrate that aldehyde-mediated adhesion to tissue strongly depends on target-tissue type and state, and propose a rational approach for the engineering of application-specific surgical sealants. Copolymeric hydrogels featuring aminated star polyethylene glycolandhigh-molecular-weight dextranaldehyde(PEG:dextran) possess a series of physico-chemical properties that can be modified to create a family of materials with tunable tissue adhesion. [14–17] The two polymer constituents of PEG:dextran were prepared as minimally viscous aqueous solutions and consistently polymerized through injection from a dual chamber syringe equipped with a mixing tip. [15,16] The cohesive integrity of PEG:dextran is derived from imine bonds that form through a Schiff base reaction between amines and aldehydes. [14–17] When crosslinked on soft-tissue surfaces, aldehydes not consumed in bulk network formation form analogous bonds with tissue amines to achieve adhesion. Aldehydes in excess of what is required for cohesion or adhesion can induce tissue toxicity. [18] Consequently, material aldehyde density is the key design parameter for informative evaluation of tissue-material adhesion and tissue response. We designed and evaluated a series of PEG:dextran formulations featuring low (8.8%, abbreviated L-PD), medium (14.0%, abbreviated M-PD), and high (20.0%, abbreviated H-PD) levels of dextran aldehyde solid content. Additional design parameters, including dextran molecular weight (10 kDa) and oxidation state (50%), and PEG amine solid content (25%) were identical among formulations, and selected to provide stable and bioreactive networks for evaluation of adhesive

Vascular Neointimal Formation and Signaling Pathway Activation in Response to Stent Injury in Insulin-Resistant and Diabetic Animals

Diabetes and insulin resistance are associated with increased disease risk and poor outcomes from cardiovascular interventions. Even drug-eluting stents exhibit reduced efficacy in patients with diabetes. We now report the first study of vascular response to stent injury in insulin-resistant and diabetic animal models. Endovascular stents were expanded in the aortae of obese insulin-resistant and type 2 diabetic Zucker rats, in streptozotocin-induced type 1 diabetic Sprague-Dawley rats, and in matched controls. Insulin-resistant rats developed thicker neointima (0.46±0.08 versus 0.37±0.06 mm2, P=0.05), with decreased lumen area (2.95±0.26 versus 3.29±0.15 mm2, P=0.03) 14 days after stenting compared with controls, but without increased vascular inflammation (ED1+ tissue macrophages). Insulin-resistant and diabetic rat vessels did exhibit markedly altered signaling pathway activation 1 and 2 weeks after stenting, with up to a 98% increase in p-ERK (anti-phospho ERK) and a 54% reduction in p-Akt (anti-phospho Akt) stained cells. Western blotting confirmed a profound effect of insulin resistance and diabetes on Akt and ERK signaling in stented segments. p-ERK/p-Akt ratio in stented segments uniquely correlated with neointimal response (R2=0.888, P=0.04) in insulin-resistant and type 1 and 2 diabetic rats, but not in lean controls. Transfemoral aortic stenting in rats provides insight into vascular responses in insulin resistance and diabetes. Shifts in ERK and Akt signaling related to insulin resistance may reflect altered tissue repair in diabetes accompanied by a shift in metabolic:proliferative balance. These findings may help explain the increased vascular morbidity in diabetes and suggest specific therapies for patients with insulin resistance and diabetes.

Endothelial Cells Provide Feedback Control for Vascular Remodeling Through a Mechanosensitive Autocrine TGF-β Signaling Pathway

Mechanical forces are potent modulators of the growth and hypertrophy of vascular cells. We examined the molecular mechanisms through which mechanical force and hypertension modulate endothelial cell regulation of vascular homeostasis. Exposure to mechanical strain increased the paracrine inhibition of vascular smooth muscle cells (VSMCs) by endothelial cells. Mechanical strain stimulated the production of perlecan and heparan sulfate glycosaminoglycans by endothelial cells. By inhibiting the expression of perlecan with an antisense vector we demonstrated that perlecan was essential to the strain-mediated effects on endothelial cell growth control. Mechanical regulation of perlecan expression in endothelial cells was governed by a mechanotransduction pathway requiring autocrine transforming growth factor β (TGF-β) signaling and intracellular signaling through the ERK pathway. Immunohistochemical staining of the aortae of spontaneously hypertensive rats demonstrated strong correlations between endothelial TGF-β, phosphorylated signaling intermediates, and arterial thickening. Further, studies on ex vivo arteries exposed to varying levels of pressure demonstrated that ERK and TGF-β signaling were required for pressure-induced upregulation of endothelial HSPG. Our findings suggest a novel feedback control mechanism in which net arterial remodeling to hemodynamic forces is controlled by a dynamic interplay between growth stimulatory signals from VSMCs and growth inhibitory signals from endothelial cells.

Thin-capped atheromata with reduced collagen content in pigs develop in coronary arterial regions exposed to persistently low endothelial shear stress.

Objective—The mechanisms promoting the focal formation of rupture-prone coronary plaques in vivo remain incompletely understood. This study tested the hypothesis that coronary regions exposed to low endothelial shear stress (ESS) favor subsequent development of collagen-poor, thin-capped plaques. Approach and Results—Coronary angiography and 3-vessel intravascular ultrasound were serially performed at 5 consecutive time points in vivo in 5 diabetic, hypercholesterolemic pigs. ESS was calculated along the course of each artery with computational fluid dynamics at all 5 time points. At follow-up, 184 arterial segments with previously identified in vivo ESS underwent histopathologic analysis. Compared with other plaque types, eccentric thin-capped atheromata developed more in segments that experienced lower ESS during their evolution. Compared with lesions with higher preceding ESS, segments persistently exposed to low ESS (<1.2 Pa) exhibited reduced intimal smooth muscle cell content; marked intimal smooth muscle cell phenotypic modulation; attenuated procollagen-I gene expression; increased gene and protein expression of the interstitial collagenases matrix-metalloproteinase-1, -8, -13, and -14; increased collagenolytic activity; reduced collagen content; and marked thinning of the fibrous cap. Conclusions—Eccentric thin-capped atheromata, lesions particularly prone to rupture, form more frequently in coronary regions exposed to low ESS throughout their evolution. By promoting an imbalance of attenuated synthesis and augmented collagen breakdown, low ESS favors the focal evolution of early lesions toward plaques with reduced collagen content and thin fibrous caps—2 critical determinants of coronary plaque vulnerability.

Heparanase Alters Arterial Structure, Mechanics, and Repair Following Endovascular Stenting in Mice

Heparan sulfate proteoglycans (HSPGs) are potent regulators of vascular remodeling and repair. Heparanase is the major enzyme capable of degrading heparan sulfate in mammalian cells. Here we examined the role of heparanase in controlling arterial structure, mechanics, and remodeling. In vitro studies supported that heparanase expression in endothelial cells serves as a negative regulator of endothelial inhibition of vascular smooth muscle cell (vSMC) proliferation. Arterial structure and remodeling to injury were also modified by heparanase expression. Transgenic mice overexpressing heparanase had increased arterial thickness, cellular density, and mechanical compliance. Endovascular stenting studies in Zucker rats demonstrated increased heparanase expression in the neointima of obese, hyperlipidemic rats in comparison to lean rats. The extent of heparanase expression within the neointima strongly correlated with the neointimal thickness following injury. To test the effects of heparanase overexpression on arterial repair, we developed a novel murine model of stent injury using small diameter self-expanding stents. Using this model, we found that increased neointimal formation and macrophage recruitment occurs in transgenic mice overexpressing heparanase. Taken together, these results support a role for heparanase in the regulation of arterial structure, mechanics, and repair.

Fluid mechanics analysis of a spring-loaded jet injector

A syringe jet injector is a device designed to administer a drug quickly and painlessly through the skin. Though syringe jet injectors have been in use for almost 50 years, current designs still suffer from inconsistent performance. To better understand the fluid mechanics of jet injection and gain insight into how the design might influence performance, two theoretical analyses to determine the fluid pressure profile at the exit orifice were conducted. The first was a continuum analysis assuming static incompressibility. Results demonstrated that the maximum jet pressure was highly sensitive to the spring constant, initial piston velocity, and piston cross-sectional area while the time to achieve the maximum pressure was most sensitive to the injection chamber length, initial piston velocity, bulk modulus of the injectant, and the piston cross-sectional area. The second analysis was a shock wave analysis. Results demonstrated a stepwise pressure-time plot that was similar in magnitude to that for the continuum analysis assuming static incompressibility. Results from these two investigations are useful for design modification of the jet injector to achieve desired pressure-time profiles at the orifice. Control of pressure-time profiles may help to achieve a more consistent and effective injection process.

Heparanase regulates thrombosis in vascular injury and stent-induced flow disturbance.

OBJECTIVES The purpose of this study was to examine the role of heparanase in controlling thrombosis following vascular injury or endovascular stenting. BACKGROUND The use of endovascular stents are a common clinical intervention for the treatment of arteries occluded due to vascular disease. Both heparin and heparan sulfate are known to be potent inhibitors of thrombosis. Heparanase is the major enzyme that degrades heparan sulfate in mammalian cells. This study examined the role of heparanase in controlling thrombosis following vascular injury and stent-induced flow disturbance. METHODS This study used mice overexpressing human heparanase and examined the time to thrombosis using a laser-induced arterial thrombosis model in combination with vascular injury. An ex vivo system was used to examine the formation of thrombus to stent-induced flow disturbance. RESULTS In the absence of vascular injury, wild type and heparanase overexpressing (HPA Tg) mice had similar times to thrombosis in a laser-induced arterial thrombosis model. However, in the presence of vascular injury, the time to thrombosis was dramatically reduced in HPA Tg mice. An ex vivo system was used to flow blood from wild type and HPA Tg mice over stents and stented arterial segments from both animal types. These studies demonstrate markedly increased thromboses on stents with blood isolated from HPA Tg mice in comparison to blood from wild type animals. We found that blood from HPA Tg animals had markedly increased thrombosis when applied to stented arterial segments from either wild type or HPA Tg mice. CONCLUSIONS Taken together, this studys results indicate that heparanase is a powerful mediator of thrombosis in the context of vascular injury and stent-induced flow disturbance.

Loss of syndecan-1 induces a pro-inflammatory phenotype in endothelial cells with a dysregulated response to atheroprotective flow.

Background: The endothelial glycocalyx extends into the arterial lumen and experiences shear forces from blood flow. Results: The loss of syndecan-1 results in a pro-inflammatory phenotype in endothelial cells with an altered response to atheroprotective flow. Conclusion: Syndecan-1 plays an important role in maintaining healthy endothelial phenotype. Significance: Therapies that retain syndecan-1 on endothelial cells may have the potential to reduce the progression of vascular disease. Fluid shear stresses are potent regulators of vascular homeostasis and powerful determinants of vascular disease progression. The glycocalyx is a layer of glycoaminoglycans, proteoglycans, and glycoproteins that lines the luminal surface of arteries. The glycocalyx interacts directly with hemodynamic forces from blood flow and, consequently, is a prime candidate for the mechanosensing of fluidic shear stresses. Here, we investigated the role of the glycocalyx component syndecan-1 (sdc-1) in controlling the shear stress-induced signaling and flow-mediated phenotypic modulation in endothelial cells. We found that knock-out of sdc-1 abolished several key early signaling events of endothelial cells in response to shear stress including the phosphorylation of Akt, the formation of a spatial gradient in paxillin phosphorylation, and the activation of RhoA. After exposure to atheroprotective flow, we found that sdc-1 knock-out endothelial cells had a phenotypic shift to an inflammatory/pro-atherosclerotic phenotype in contrast to the atheroprotective phenotype of wild type cells. Consistent with these findings, we found increased leukocyte adhesion to sdc-1 knock-out endothelial cells in vitro that was reduced by re-expression of sdc-1. In vivo, we found increased leukocyte recruitment and vascular permeability/inflammation in sdc-1 knock-out mice. Taken together, our studies support a key role for sdc-1 in endothelial mechanosensing and regulation of endothelial phenotype.